The goal of this project is to identify molecular mechanisms involved in the regulation of beta-adrenergic receptor (beta-AR)-coupled adenylylcyclase. We previously reported that human beta1AR and beta2AR are regulated differently by agonists whether the receptors are endogenously expressed in human cell lines or stably expressed in transfected hamster cell lines. When exposed to agonist, the cells expressing beta2AR exhibit a rapid, typical pattern of desensitization of agonist-stimulated adenylylcyclase. Both maximum stimulation (V/max) is reduced and dose response (K/act) is shifted to lower sensitivity. By contrast, agonist-treated cells expressing beta1AR display a shift in K/act, but little or no reduction in V/max. It has been shown that desensitization of the beta2AR is mediated by protein kinase A (PKA) via phosphorylation of the third intracellular loop of the receptor and by the beta-adrenergic receptor kinase (betaARK) via phosphorylation of the C-terminus. By using specific kinase inhibitors, we have found that the reduction in V/max was mediated by betaARK. This included the modest reduction observed for beta1AR. The shift in K/act was mediated by PKA. This was further confirmed by exposing the cells to a cAMP derivative. With cells expressing either subtype, there was a shift in K/act, but no reduction in V/max. These results indicated that human beta1AR was resistant to betaARK-mediated desensitization. By contrast, hamster cells expressing rat beta1AR displayed a pattern of agonist-mediated desensitization similar to that of cells expressing human beta2AR. Whereas human beta1AR and beta2AR are only 54% homologous, the human and rat beta1AR are 91% homologous. Thus, only a small region of the receptor may be involved in betaARK-mediated desensitization.